Structural basis for cyclodextrins' suppression of human growth hormone aggregation

Area of Science:

• Biochemistry
• Protein Chemistry
• Physical Chemistry

Background:

• Therapeutic proteins often require room temperature storage, risking aggregation and functional loss.
• Cyclodextrins (CDs) are known protein aggregation suppressors, particularly interacting with aromatic amino acids.
• Detailed structural insights into CD-protein interactions, especially at low pH, are lacking.

Purpose of the Study:

• To investigate the structural interactions between human growth hormone (hGH) and various cyclodextrins (CDs) at low pH.
• To elucidate the mechanism by which CDs prevent hGH aggregation under destabilizing conditions.

Main Methods:

• Stopped-flow kinetics to monitor aggregation reactions in the presence of different CDs.
• Fluorescence-based titration to determine binding affinities.
• Nuclear Magnetic Resonance (NMR) spectroscopy to analyze structural changes and binding sites.

Main Results:

• Beta-cyclodextrin (β-CD) derivatives effectively prevented hGH aggregation at pH 2.5, while alpha- and gamma-CDs were less potent.
• Apparent dissociation constant for hGH-β-CD interaction was determined to be approximately 6 mM.
• NMR studies revealed that β-CD promotes a more unfolded hGH conformation at low pH and binds to aromatic side chains.

Conclusions:

• Beta-cyclodextrins are potent stabilizers of human growth hormone against aggregation at low pH.
• The mechanism involves β-CD binding to aromatic amino acids, which are key components of aggregation-nucleating regions.
• These findings provide structural basis for using CDs as excipients for therapeutic protein stabilization.